Figure 1
From: Monoclonal regulatory T cells provide insights into T cell suppression
(a) Representative flow cytometry plots of CD4+ LN T cells from B3K506TCR Tg Rag−/− (B3K506 Tconv) and B3K506 TCR Tg Foxp3 Tg Rag−/− (B3K506 Treg) mice are shown. Numbers in plots depict % of cells in the quadrant. (b) Representative histograms show surface marker expression on monoclonal LN B3K506 Tregs (
), polyclonal B6 Foxp3EGFP Tregs (
), polyclonal B6 Tconvs () and monoclonal LN B3K506 Tconvs (
), n = 4. (c) In vitro anti-CD3ε suppression assays. Representative proliferation of polyclonal B6 CD4+ Tconvs co-cultured with polyclonal B6 Foxp3EGFP Tregs (
), monoclonal B3K506 Tregs (
) or B3K506 Tconv (
) at a 1Tconv/4Treg ratio. Grey histograms indicate proliferation of B6 CD4+ Tconvs cultured without Tregs. Numbers in histograms depict % proliferated cells. Graph shows mean % suppression + /− SD at 72h, n = 5. (d) In vitro anti-CD3ε suppression assays. Capacity of monoclonal B3K506 Tregs (
), polyclonal B6 Foxp3EGFP Tregs (
) and monoclonal B3K506 Tconv (negative control; 
) to suppress proliferation of polyclonal B6 CD4+ T cells at varying Treg/Tconv ratios. Data show mean % suppression + /− SD, n = 3. (e/f) Antigen stimulated suppression assays. (e) CFSE labelled OT-II Tconvs were co-cultured with B3K506 Tregs at a 1Tconv/4Treg ratio in the presence (
) or absence (
) of 3K peptide, or with B3K506 Tconv (
) in the presence of 3K peptide. All cultures contained OVA peptide. OT-II Tconv proliferation in the absence of Tregs is shown (
). Numbers in histograms represent % proliferated cells. (f) OT-II Tconv proliferation index and % suppression in co-cultures described in E). Monoclonal B3K506 Tregs + 3K peptide (
); monoclonal B3K506 Tregs −3K peptide (
); monoclonal B3K506 Tconvs (
); OT-II Tconvs alone (
). All cultures contained OVA peptide. Data show mean proliferation index + /− SD, n = 9–12. Statistical significance was calculated using a one-way ANOVA and subsequently Bonferroni’s multiple comparisons test. NS = p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
